symbiosis growth analysis
Analysis - David J. Weston
Experimentation - Colleen Husley & Megan Gable
11/16/2018
1 Introduction
The symbiosis between Sphagnum mosses and cyanobacteria has long been recognized (Limpricht, 1890) and has been implicated in improved moss growth. This together with evidence form other plant - cyanobacteria symbioses suggest that this is not just a symbiosis (interacting species), but perhaps a mutualsitc interaction – thereby benefiting both interacting speices. To investigate this further, Colleen Hulsey and Megan Gable conducted a numer of experiments to investigate the growth consequences of cyanobacteria and sphagnum symbiosis on growth patterns at differing pH. conditions.
2 Growth 2018 symbiosis approach and measurements
*Experimental conditions & design +conducted on 08/09/2018 +plants pH nitrogen depleted for 2 weeks prior to start +experiment ran for 2 weeks +condition 1: Nitrogen (N), 6 replicates -N, 2 replicates for low N (5mL/L NaNO3– Mm?) +Condition 2: pH, 3.5, 5.5, 8.5 +speices: Sphagnum fallax MN alone, Nostoc muscorum 1037 alone, both species together +amounts: 1 moss capitulum, 0.1g cyanobacteria +3 - 12 well plates, 2 ml liquid medium per well.
*Measurements +fluorcam: area_mm2, QY_max (initial, weekly, final) +pH initial, pH weekly, pH final +weight beginning and end after water removed with centrifugation
*tests +simple scatter and boxplots to view realtionship
3 Growth 2018 data import and summary
library(tidyverse)
#knitr by default will set the working directory to the source file location
#setwd("~/Documents/Manuscripts:data/Symbiosis_study_2018")
dat1 <- read_csv("fallax_cyano_symbiosis_colleen_10_01_2018.csv") %>% mutate(delta_weight = delta_weight_g *1000)
names(dat1)## [1] "Sample_well" "component_measured" "condition"
## [4] "initial_pH" "final_pH" "initial_weight_g"
## [7] "final_weight_g" "delta_weight_g" "delta_weight"summary(dat1)## Sample_well component_measured condition initial_pH
## Length:72 Length:72 Length:72 Min. :3.500
## Class :character Class :character Class :character 1st Qu.:3.500
## Mode :character Mode :character Mode :character Median :5.500
## Mean :5.833
## 3rd Qu.:8.500
## Max. :8.500
##
## final_pH initial_weight_g final_weight_g delta_weight_g
## Min. :3.700 Min. :0.00140 Min. :0.01430 Min. :-0.03560
## 1st Qu.:5.800 1st Qu.:0.01342 1st Qu.:0.04083 1st Qu.: 0.01082
## Median :6.200 Median :0.09345 Median :0.09570 Median : 0.03030
## Mean :6.117 Mean :0.06677 Mean :0.10127 Mean : 0.03766
## 3rd Qu.:6.700 3rd Qu.:0.10622 3rd Qu.:0.15368 3rd Qu.: 0.06940
## Max. :7.440 Max. :0.12040 Max. :0.22060 Max. : 0.11090
## NA's :12 NA's :12 NA's :12
## delta_weight
## Min. :-35.60
## 1st Qu.: 10.82
## Median : 30.30
## Mean : 37.66
## 3rd Qu.: 69.40
## Max. :110.90
## dim(dat1)## [1] 72 94 Growth 2018 data boxplot analysis
- Delta growth (weight) by organism regardless of pH; 2. Delta growth by organism and by pH; 3. Delta growth for entier wel by pH
Fig. 1: 2018 data, change in growth (initial weight (wt) - final wt among the two symbiotic partners alone and when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Cyano in symbiosis includes, endophytes, epiphytes and free living cyano. Data incuded all pH conditions
Fig. 2: 2018 data, change in growth among interacting partners faceted by starting pH condiiton. Measured components as described in fig. 1
Fig. 3: 2018 data, total well biomass change from experiment start to finish. Includes moss + cyano endophyte and free living cyanobacteria not attached to plant
## Warning: Removed 12 rows containing non-finite values (stat_boxplot).## Warning: Removed 12 rows containing missing values (geom_point).
Fig. 4: 2018 data, change in pH change as measured by (final - initial values)
## Warning: Removed 12 rows containing non-finite values (stat_boxplot).## Warning: Removed 12 rows containing missing values (geom_point).
Fig. 5: 2018 data, absolute value of pH change as measured by (final - initial values)
5 Growth 2017 symbiosis approach and measurements
need experimental details here…
6 Growth 2017 data import and summary
dat2 <- read_csv("fallax_cyano_symbiosis_allyssa_colleen_2017.csv")
dat3 <- dat2 %>% filter(pH == 3.5 | pH == 5.5 | pH == 8.5)
names(dat3)## [1] "condition" "replicate" "pH" "plate"
## [5] "well" "weight_initial" "weight_final" "delta_weight"summary(dat3)## condition replicate pH plate
## Length:72 Min. :1.0 Min. :3.500 Min. :1
## Class :character 1st Qu.:2.0 1st Qu.:3.500 1st Qu.:3
## Mode :character Median :3.5 Median :5.500 Median :5
## Mean :3.5 Mean :5.833 Mean :5
## 3rd Qu.:5.0 3rd Qu.:8.500 3rd Qu.:7
## Max. :6.0 Max. :8.500 Max. :9
## NA's :18 NA's :18
## well weight_initial weight_final delta_weight
## Min. : 1.000 Min. : 3.11 Min. : 8.67 Min. :-21.330
## 1st Qu.: 3.000 1st Qu.:21.73 1st Qu.: 29.20 1st Qu.: 8.562
## Median : 6.500 Median :30.00 Median : 46.91 Median : 23.870
## Mean : 6.333 Mean :33.72 Mean : 64.98 Mean : 32.358
## 3rd Qu.: 9.000 3rd Qu.:40.02 3rd Qu.: 96.84 3rd Qu.: 46.220
## Max. :12.000 Max. :87.22 Max. :177.43 Max. :127.830
## NA's :18 NA's :18 NA's :18dim(dat3)## [1] 72 87 Boxplot analysis of 2017 data
Fig. 6: 2017 data, change in growth (initial weight (wt) - final wt among the two symbiotic partners alone, when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Conditions aggregated over pH
Fig. 7: 2017 data, change in growth (initial weight (wt) - final wt among the two symbiotic partners alone, when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Similar to Fig. 6 except faceted by pH
Fig. 8: Combined 2018 and 2017 data for change in growth (initial weight (wt) - among the two symbiotic partners alone, when added togather but grown seperately (_seperate) and when grown together in same well (_symbiosis). Data is faceted by pH
8 ANOVA analysis of 2017 and 2018 data.
Specifically testing if cyano and moss grown alone, increased in growth at a different rate than when grown together in smbiosis.
## Df Sum Sq Mean Sq F value Pr(>F)
## condition 3 20854 6951 9.251 4.05e-05 ***
## pH 2 8810 4405 5.862 0.00473 **
## condition:pH 6 2464 411 0.546 0.77075
## Residuals 60 45086 751
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1computing summary stats
## # A tibble: 12 x 5
## # Groups: condition [?]
## condition pH count mean sd
## <fct> <fct> <int> <dbl> <dbl>
## 1 cyano_alone_2017 pH_3.5 6 1.38 9.17
## 2 cyano_alone_2017 pH_5.5 6 5.64 16.4
## 3 cyano_alone_2017 pH_8.5 6 29.8 39.4
## 4 moss_alone_2017 pH_3.5 6 13.8 14.0
## 5 moss_alone_2017 pH_5.5 6 32.1 26.2
## 6 moss_alone_2017 pH_8.5 6 24.2 15.4
## 7 Cyanobacteria_moss_seperate_2017 pH_3.5 6 15.2 5.52
## 8 Cyanobacteria_moss_seperate_2017 pH_5.5 6 37.7 37.7
## 9 Cyanobacteria_moss_seperate_2017 pH_8.5 6 54.0 51.4
## 10 Cyanobacteria_moss_symbiosis_2017 pH_3.5 6 44.1 20.1
## 11 Cyanobacteria_moss_symbiosis_2017 pH_5.5 6 55.5 23.3
## 12 Cyanobacteria_moss_symbiosis_2017 pH_8.5 6 74.8 30.7mean seperation iwth Tukeys
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = delta_weight ~ condition + pH + condition:pH, data = dat.2017)
##
## $condition
## diff
## moss_alone_2017-cyano_alone_2017 11.11278
## Cyanobacteria_moss_seperate_2017-cyano_alone_2017 23.38444
## Cyanobacteria_moss_symbiosis_2017-cyano_alone_2017 45.84889
## Cyanobacteria_moss_seperate_2017-moss_alone_2017 12.27167
## Cyanobacteria_moss_symbiosis_2017-moss_alone_2017 34.73611
## Cyanobacteria_moss_symbiosis_2017-Cyanobacteria_moss_seperate_2017 22.46444
## lwr
## moss_alone_2017-cyano_alone_2017 -13.033058
## Cyanobacteria_moss_seperate_2017-cyano_alone_2017 -0.761391
## Cyanobacteria_moss_symbiosis_2017-cyano_alone_2017 21.703053
## Cyanobacteria_moss_seperate_2017-moss_alone_2017 -11.874169
## Cyanobacteria_moss_symbiosis_2017-moss_alone_2017 10.590276
## Cyanobacteria_moss_symbiosis_2017-Cyanobacteria_moss_seperate_2017 -1.681391
## upr
## moss_alone_2017-cyano_alone_2017 35.25861
## Cyanobacteria_moss_seperate_2017-cyano_alone_2017 47.53028
## Cyanobacteria_moss_symbiosis_2017-cyano_alone_2017 69.99472
## Cyanobacteria_moss_seperate_2017-moss_alone_2017 36.41750
## Cyanobacteria_moss_symbiosis_2017-moss_alone_2017 58.88195
## Cyanobacteria_moss_symbiosis_2017-Cyanobacteria_moss_seperate_2017 46.61028
## p adj
## moss_alone_2017-cyano_alone_2017 0.6191255
## Cyanobacteria_moss_seperate_2017-cyano_alone_2017 0.0610500
## Cyanobacteria_moss_symbiosis_2017-cyano_alone_2017 0.0000289
## Cyanobacteria_moss_seperate_2017-moss_alone_2017 0.5396394
## Cyanobacteria_moss_symbiosis_2017-moss_alone_2017 0.0018777
## Cyanobacteria_moss_symbiosis_2017-Cyanobacteria_moss_seperate_2017 0.0771172resdiual check
##
## Shapiro-Wilk normality test
##
## data: aov_residuals
## W = 0.96343, p-value = 0.03459Starting the 2018 data
## Df Sum Sq Mean Sq F value Pr(>F)
## condition 3 11694 3898 5.216 0.00429 **
## pH 2 32248 16124 21.577 6.93e-07 ***
## condition:pH 6 11846 1974 2.642 0.03159 *
## Residuals 36 26902 747
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1computing summary stats for 2018
## # A tibble: 12 x 5
## # Groups: condition [?]
## condition pH count mean sd
## <fct> <fct> <int> <dbl> <dbl>
## 1 moss_alone_2018 pH_3.5 4 32.6 31.4
## 2 moss_alone_2018 pH_5.5 4 35.5 16.4
## 3 moss_alone_2018 pH_8.5 4 29.8 11.5
## 4 cyano_alone_2018 pH_3.5 4 -21.4 24.9
## 5 cyano_alone_2018 pH_5.5 4 20.5 28.6
## 6 cyano_alone_2018 pH_8.5 4 63.2 23.0
## 7 Cyanobacteria_moss_symbiosis_2018 pH_3.5 4 14.3 21.5
## 8 Cyanobacteria_moss_symbiosis_2018 pH_5.5 4 59.6 42.4
## 9 Cyanobacteria_moss_symbiosis_2018 pH_8.5 4 105. 5.81
## 10 Cyanobacteria_moss_seperate_2018 pH_3.5 4 11.3 41.3
## 11 Cyanobacteria_moss_seperate_2018 pH_5.5 4 56.0 36.3
## 12 Cyanobacteria_moss_seperate_2018 pH_8.5 4 93.0 16.9mean seperation with Tukeys for 2018
## Tukey multiple comparisons of means
## 95% family-wise confidence level
##
## Fit: aov(formula = delta_weight ~ condition + pH + condition:pH, data = dat.2018)
##
## $condition
## diff
## cyano_alone_2018-moss_alone_2018 -11.858333
## Cyanobacteria_moss_symbiosis_2018-moss_alone_2018 26.866667
## Cyanobacteria_moss_seperate_2018-moss_alone_2018 20.800000
## Cyanobacteria_moss_symbiosis_2018-cyano_alone_2018 38.725000
## Cyanobacteria_moss_seperate_2018-cyano_alone_2018 32.658333
## Cyanobacteria_moss_seperate_2018-Cyanobacteria_moss_symbiosis_2018 -6.066667
## lwr
## cyano_alone_2018-moss_alone_2018 -41.915110
## Cyanobacteria_moss_symbiosis_2018-moss_alone_2018 -3.190110
## Cyanobacteria_moss_seperate_2018-moss_alone_2018 -9.256777
## Cyanobacteria_moss_symbiosis_2018-cyano_alone_2018 8.668223
## Cyanobacteria_moss_seperate_2018-cyano_alone_2018 2.601556
## Cyanobacteria_moss_seperate_2018-Cyanobacteria_moss_symbiosis_2018 -36.123444
## upr
## cyano_alone_2018-moss_alone_2018 18.19844
## Cyanobacteria_moss_symbiosis_2018-moss_alone_2018 56.92344
## Cyanobacteria_moss_seperate_2018-moss_alone_2018 50.85678
## Cyanobacteria_moss_symbiosis_2018-cyano_alone_2018 68.78178
## Cyanobacteria_moss_seperate_2018-cyano_alone_2018 62.71511
## Cyanobacteria_moss_seperate_2018-Cyanobacteria_moss_symbiosis_2018 23.99011
## p adj
## cyano_alone_2018-moss_alone_2018 0.7140984
## Cyanobacteria_moss_symbiosis_2018-moss_alone_2018 0.0937375
## Cyanobacteria_moss_seperate_2018-moss_alone_2018 0.2614148
## Cyanobacteria_moss_symbiosis_2018-cyano_alone_2018 0.0071606
## Cyanobacteria_moss_seperate_2018-cyano_alone_2018 0.0288313
## Cyanobacteria_moss_seperate_2018-Cyanobacteria_moss_symbiosis_2018 0.9476697resdiual check 2018 data
Shapiro-Wilk test for normality
##
## Shapiro-Wilk normality test
##
## data: aov_residuals
## W = 0.96343, p-value = 0.03459#Levene's Test for homogenity of variances
library(car)## Loading required package: carData##
## Attaching package: 'car'## The following object is masked from 'package:dplyr':
##
## recode## The following object is masked from 'package:purrr':
##
## someleveneTest(delta_weight ~ condition, data = dat.2018)## Levene's Test for Homogeneity of Variance (center = median)
## Df F value Pr(>F)
## group 3 3.7312 0.01787 *
## 44
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1leveneTest(delta_weight ~ pH, data = dat.2018)## Levene's Test for Homogeneity of Variance (center = median)
## Df F value Pr(>F)
## group 2 0.0209 0.9793
## 45